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2459
Development 129, 2459-2472 (2002)
Printed in Great Britain © The Company of Biologists Limited 2002
DEV9785
BMP signaling patterns the dorsal and intermediate neural tube via regulation
of homeobox and helix-loop-helix transcription factors
John R. Timmer, Charlotte Wang and Lee Niswander*
Molecular Biology Program and Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021,
USA
*Author for correspondence (e-mail: [email protected])
Accepted 19 February 2002
SUMMARY
In the spinal neural tube, populations of neuronal
precursors that express a unique combination of
transcription factors give rise to specific classes of neurons
at precise locations along the dorsoventral axis.
Understanding the patterning mechanisms that generate
restricted gene expression along the dorsoventral axis is
therefore crucial to understanding the creation of diverse
neural cell types. Bone morphogenetic proteins (BMPs) and
other transforming growth factor β (TGFβ) proteins are
expressed by the dorsal-most cells of the neural tube (the
roofplate) and surrounding tissues, and evidence indicates
that they play a role in assigning cell identity. We have
manipulated the level of BMP signaling in the chicken
neural tube to show that BMPs provide patterning
information to both dorsal and intermediate cells. BMP
regulation of the expression boundaries of the homeobox
proteins Pax6, Dbx2 and Msx1 generates precursor
populations with distinct developmental potentials. Within
the resulting populations, thresholds of BMP act to set
expression domain boundaries of developmental regulators
of the homeobox and basic helix-loop-helix (bHLH)
families, ultimately leading to the generation of a diversity
of differentiated neural cell types. This evidence strongly
suggests that BMPs are the key regulators of dorsal cell
identity in the spinal neural tube.
INTRODUCTION
subdivision of these regions is crucial to an understanding of
neural patterning. There is substantial evidence that the sonic
hedgehog protein (SHH) controls both regional identity and
cell fate in the ventral neural tube. Elimination of Shh gene
function results in severe abnormalities in the spinal neural
tube, including the failure to generate many ventral cell types
(Chiang et al., 1996; Ericson et al., 1996; Marti et al., 1995).
Activation of the Shh signaling pathway in dorsal neural tissue
blocks the expression of dorsally restricted proteins and
imposes a ventral fate on dorsal cells (Epstein et al., 1996;
Hynes et al., 2000; Roelink et al., 1995). In addition to
providing a ventral identity to cells in the developing neural
tube, SHH has been shown to participate in the division of the
ventral neural tube into discrete populations of ventral neuronal
precursors (Briscoe et al., 2000). SHH acts to generate a
morphogen field in the ventral neural tube, and several
transcriptional regulators of the homeobox gene family have
expression boundaries set by specific thresholds of SHH
signaling (Ericson et al., 1997a; Ericson et al., 1997b). As a
result, small populations of proliferating neuronal precursors
express specific combinations of homeobox proteins; each
population then gives rise to a single class of differentiated
neurons.
It has been suggested that members of the TGFβ superfamily,
including several of the BMPs, play a similar role in the dorsal
neural tube (Lee and Jessell, 1999). BMP gene expression begins
Patterning information along the dorsoventral axis of the spinal
neural tube can be viewed as being generated by two processes:
the assignment of regional identity and the division of these
regions into discrete domains of gene expression. The early
expression of developmental regulators by broad areas of the
neural tube suggests that regional identity acts to restrict the
potential fates adopted by cells within that region. Although
the expression of individual genes may act to mark a particular
region, multiple factors may contribute to the establishment
and maintenance of regional identity over the course of neural
tube development. Large regions in the neural tube, however,
give rise to many classes of terminally differentiated neurons,
suggesting that these regions are subdivided into discrete cell
populations. Overlapping and exclusive expression of specific
combinations of developmental regulators within a given
region has been shown to generate discrete populations
(Briscoe et al., 2000; Ericson et al., 1996; Pierani et al., 1999).
Although the regionalization and subdivision of the neural tube
can be viewed as separate processes, there can clearly be some
overlap between them. Pax3 and Pax7, which are believed to
contribute to a dorsal identity, also act to subdivide a
population of intermediate cells into two distinct populations
(Mansouri and Gruss, 1998; Pierani et al., 1999).
Understanding the generation of regional identity and the
Key words: Neural tube, Bone morphogenetic protein, Dorsal-ventral
patterning, Basic Helix-Loop-Helix, Homeobox, Chick
2460 J. R. Timmer, C. Wang and L. Niswander
in tissue juxtaposed with the dorsal neural tube prior to neural
tube closure. After closure of the neural tube, several TGFβ
superfamily members are expressed by the roofplate and
surrounding tissues (Basler et al., 1993; Lee et al., 1998; Liem
et al., 1995). Moreover, exposure of cultured explants of
undifferentiated neural tissue to BMPs elicits changes in the
expression of many developmental regulators known to be
expressed by cells of the dorsal neural tube (Lee et al., 1998;
Liem et al., 1997; Pierani et al., 1999). These experiments have
also indicated that the expression of several of these genes is
sensitive to the concentration of BMPs, suggesting that BMPs
can also act as morphogens in generating patterning information.
In intact embryonic neural tissue, however, the activity of BMPs
is likely to be influenced by the expression of BMP antagonists
and ligands of other signaling pathways, both by surrounding
tissues and within the neural tube itself (Bertrand et al., 2000;
Ericson et al., 1995; Hollyday et al., 1995; Liem et al., 2000;
McMahon et al., 1998; Pierani et al., 1999; Pituello et al., 1995;
Williams et al., 1995). In addition, the Shh signaling pathway
has also been shown to both influence the expression of BMPresponsive genes (Liem et al., 1997; Liem et al., 1995; Selleck
et al., 1998) and to be influenced by BMP signaling(Liem et al.,
2000). Thus, there remains a need to identify the role of BMP
signaling in its proper context.
Clarifying the precise role of BMP signaling in neural tissue
in vivo has proven difficult. The large number of TGFβ ligands
expressed in neural tissue suggests a degree of functional
redundancy, and gene knockout experiments targeting the
BMPs or their receptors in mice have in large part generated
mice that either lacked a neural phenotype or died at early
stages of development (Beppu et al., 2000; Dudley and
Robertson, 1997; Dunn et al., 1997; Luo et al., 1995; Solloway
et al., 1998; Yi et al., 2000). The single exception, the GDF-7
knockout, affects only a subpopulation of the neurons
generated from the dorsal-most population of neural precursors
(Lee et al., 1998). Elimination of the roofplate genetically
(Millonig et al., 2000) or via the roofplate-specific expression
of a toxin (Lee et al., 2000) has supported a role for the
roofplate and the BMPs it expresses in generating some dorsal
neural cell types. These experiments, however, differ in the
severity of their effects on gene expression, possibly as a result
of either differences in the timing of roofplate elimination or
due to alteration of surrounding tissues, which express TGFβ
ligands or other factors.
We have taken an opposite approach to study the role of
BMPs in patterning the neural tube. We have activated the
BMP signaling pathway within cells of the developing neural
tube in a cell autonomous manner via the use of mutated BMP
receptor (BMPR) constructs that are active in the absence of
ligand. We have found that BMP signaling regulates patterning
genes that promote regional identity, as well as those that
divide these regions into discrete cell populations. Our results
also suggest that thresholds of BMP signaling activity regulate
the proteins that ultimately generate a diversity of dorsal
neuronal cell types.
MATERIALS AND METHODS
Embryo manipulations
Fertilized White Leghorn eggs were obtained from SPAFAS
(Norwich, Conn.) and incubated at 39°C. Two methods were used to
express the activated BMPR and other proteins in the developing
chicken neural tube: retroviral infection (Fekete and Cepko, 1993) and
electroporation (Muramatsu et al., 1997). In both cases, procedures
were timed so that construct expression would begin shortly after
neural tube closure in the caudal regions of the embryo. Viral
infections were carried out at Hamburger and Hamilton (Hamburger
and Hamilton, 1992) stage 10-12 and electroporations were performed
at stages 14-16. Embryos were dissected approximately 48 hours after
these manipulations, at stages 22-24, fixed in 4% parafomaldehyde in
PBS for 1 hour, washed extensively in PBS, embedded in OCT, and
cryosectioned at 10 µm. Sections were taken from the region between
the fore- and hind-limbs. All results described here were obtained in
at least four embryos.
Purified, replication-competent retroviruses were injected directly
into the lumen of the neural tube. Viral infection was assayed via
immunofluorescence using an antibody to the viral gag protein, as
shown in Fig. 1A. For electroporations, DNA was injected into the
lumen of the neural tube and introduced into cells by three pulses of
25 mV (50 ms each). Electroporated cells were marked by the coelectroporation of a vector expressing EGFP (Clontech, for example,
see inset of Fig. 1C). Cell autonomy was correlated with phenotype
in electroporated samples; virally infected samples generally had
uniform expression of viral antigen, owing to the continual spread of
RCAS. In some samples electroporated with activated BMPR-Ib,
extensive construct expression resulted in apoptosis, disrupting the
morphology of the neural tube (data not shown). Electroporated
samples shown here (except Fig. 1H) had limited expression and no
phenotype indicative of apoptosis.
BMPR and homeobox expression constructs
Point mutations have been generated in both the type Ia and Ib BMPRs
that mimic ligand driven phosphorylation and cause these receptors to
be active in the absence of ligands (Wieser et al., 1995). BMPR
constructs were expressed from either pMiW III (Muramatsu et al.,
1997), pCAGGS (Niwa et al., 1991) or the Clontech EGFP-N1 vector
(CMV promoter; GFP coding region was replaced by that of activated
BMPR-IB). Levels of expression driven by these vectors correlated with
the strength of phenotypes as detailed in Table 1. All samples shown
here were generated with activated BMPR-Ib (except Fig. 1K,L).
RCAS viruses expressing activated BMPR-Ib (Zou et al., 1997),
Msx1 (Bendall et al., 1999) or Dbx2 (Pierani et al., 1999) have been
described. RCAS-Msx1 infection was carried out solely by viral
injection; RCAS-Dbx2 infection was carried out both by viral
injection and by electroporation of viral DNA. No difference in the
results generated by the two methods was apparent.
Protein and transcript detection
Immunofluorescent detection of proteins on cryosections was carried
Table 1
Construct
Activity
Assays
PMiwIII-caBMPR-Ib
pCAGGS-caBMPR-Ia
RCAS-caBMPR-Ib
pCMV-caBMPR-Ib
+++++
++++
++/–*
+
1,2,3,5,6
1,2,4,5,6
1,2,3
1,2,4,5
Assays: (1) induction of Msx protein expression in its endogenous domain;
(2) induction of Msx protein expression in ventral cells; (3) induction of
apoptosis (measured by TUNEL), resulting in altered neural tube
morphology; (4) generation of a morphological change similar to that in assay
3; (5) expression of construct transcript (higher level of expression correlates
with higher construct activity); (6) higher activity of caBMPR-Ib versus
caBMPR-Ia was also seen in other tissues (Zhou, 1995).
*Activity in infected cells varied according to the timing of infection and
viral spread.
BMP regulation of neural tube patterning 2461
out using standard methods (Yamada et al., 1993). The following
antibodies were obtained from the Developmental Studies Hybridoma
Bank (the Developmental Studies Hybridoma Bank was developed
under the auspices of the NICHD and maintained by The University
of Iowa, Department of Biological Sciences, Iowa City, IA 52242):
AMV viral gag protein (Potts et al., 1987), En1 (Ericson et al., 1997b),
Isl1 (Ericson et al., 1992), Lim1/2 (Tsuchida et al., 1994), Msx (Liem
et al., 1995), Pax6 (Ericson et al., 1997b) and Pax7 (Ericson et al.,
1996). Dbx1, Dbx2, Evx1 (Pierani et al., 1999), and LH2A/B (Lee et
al., 1998) have been described previously and were kindly provided
by Tom Jessell. Mash1 (Horton et al., 1999) and Math1 (Helms and
Johnson, 1998) were kindly provided by Jane Johnson. Species
appropriate secondary antibodies conjugated to either FITC or Cy3
were obtained from Jackson ImmunoResearch.
In situ hybridization on cryosections was performed using
established procedures (Holmes and Niswander, 2001). Probes for
Ngn1 and Ngn2 (Perez et al., 1999) were a gift from D. Anderson.
Probes for Msx1 and Msx2 (Chan-Thomas et al., 1993) were a gift
from B. Robert.
RESULTS
An activated BMPR was expressed in the developing chicken
neural tube after neural tube closure, either by infection with
a modified retrovirus (RCAS; transfected cells were identified
by expression of a viral protein; see Fig. 1A) or via
Fig. 1. BMP signaling regulates Pax gene expression in dorsal and intermediate domains. (A) Identification of virally infected cells by
immunofluorescence using an antibody to the viral gag protein. (B-E) Electroporation constructs generate different levels of BMP signaling.
Electroporations into the left side of the neural tube included a GFP expression plasmid to mark transfected cells. (B,C) Low levels of activated
BMPR expression generate weak BMP target gene activation. (B) Expression of activated BMPR-Ib driven by the EGFP vector is barely
detectable over endogenous expression of BMPR-Ib (stage 24). An alternate section (C) shows correspondingly weak activation of Msx protein
expression, a target of BMP signaling. Inset shows GFP, which labels transfected cells, indicating that the construct is present. (D,E) High
levels of activated BMPR expression generate strong BMP target gene activation. (D) Expression of activated BMPR-Ib driven by pMiW-III
vector results in expression readily detected above the endogenous expression pattern (stage 24 embryo, detection reaction stopped prior to
clear detection of endogenous BMPR-Ib expression). This results in a correspondingly robust MSX response in alternate sections (E).
(F-H) Pax7 expression is activated by BMP signaling. (F) Wild-type Pax7 expression in a stage 23 embryo. Ectopic Pax7 expression
(arrowheads) is apparent in virally infected (G, stage 24) and electroporated (H, stage 24) embryos. The unusual morphology in H was caused
by apoptosis (see Materials and Methods). (I-M) Expression of Pax6 is regulated by BMP signaling. The vertical bar denotes the normal region
of high-level Pax6 expression in the intermediate region of the neural tube. (I) Wild-type Pax6 expression in a stage 24 sample. (J) Pax6
expression is repressed by high levels of BMP signaling (arrowheads; inset shows that Pax6 repression is limited to transfected cells). High
signaling was generated by electroporation of pMiWIII-activated BMPR-Ib; sample is stage 24. (K,L) High level expression of Pax6 in
intermediate cells is downregulated by moderate levels of BMP signaling. Moderate signaling was generated by electroporation of pCAGGSactivated BMPR-Ia; sample is stage 24, insets show correlation of alterations with transfected cells. (M) Ventral Pax6 expression is upregulated
in response to BMP signaling (arrowhead; viral infection, stage 23).
2462 J. R. Timmer, C. Wang and L. Niswander
electroporation of expression plasmids (transfected cells were
marked by co-electroporation of a GFP expression plasmid; see
inset in Fig. 1C). The identification of transfected cells
indicated that changes in gene expression generated by the
activated receptor appear to be cell autonomous (see insets of
all electroporated and some viral samples). No qualitative
difference between receptors Ia and Ib was apparent; however,
quantitative differences could be elicited depending on the
choice of receptor and vector used. Accordingly, for some
experiments we have generated different levels of BMP
signaling as indicated by Table 1 and Fig. 1B-E. Low levels of
BMP signaling were generated using a construct that drove
weak construct expression (Fig. 1B; correspondingly weak
activation of BMP target gene Msx shown in Fig. 1C). High
levels of signaling were generated using a stronger expression
vector (Fig. 1D; extensive activation of Msx shown in Fig. 1E).
Transfection of any of these constructs did not appear to alter
the pattern of proliferation in undifferentiated neural cells (data
not shown). Although the strongest two constructs could
induce apoptosis in neural cells, the apoptosis was unlocalized
(data not shown), indicating that alterations in patterning were
not mediated by selective cell death. Samples shown here did
not have a morphology consistent with significant apoptosis
(excepting Fig. 1H).
BMP signaling regulates Pax gene expression
Some of the earliest indicators of dorsoventral patterning in the
spinal neural tube are genes of the Pax family (Chalepakis et
al., 1993). In wild-type embryos, expression of Pax7 and the
related Pax3 gene is restricted to dorsal cells, prior to closure
of the neural tube, at least partly due to repression by SHH
(Goulding et al., 1993; Hynes et al., 2000; Liem et al., 1995).
At stage 23, expression of Pax7 in untransfected embryos
occurs only in cells within the dorsal half of the neural tube
(Fig. 1F). As seen in Fig. 1G,H, expression of activated BMPR
results in the expression of Pax7 in ventral neural tissue.
Ventral Pax7 expression occurred most frequently in those cells
in proximity to the normal expression boundary, although
Pax7+ cells were detected further ventrally, including in close
proximity to the floorplate (arrowheads in Fig. 1G). In
electroporated samples, ventral expression of Pax7 was more
frequent and robust (Fig. 1H). These data indicate that BMP
signaling can promote the expression of Pax7 in ventral cells,
but that activation may be limited in embryos with lower levels
of BMP signaling. This limited activation may be due to
reduced competence of ventral cells to express Pax7, or the
presence of other factors, such as SHH, that repress Pax7
expression.
The regulation of Pax6 expression is more complex. Pax6
mRNA is first detectable immediately after neural tube closure
(Ericson et al., 1997b; Walther and Gruss, 1991). Ventrally,
Pax6 has been shown to respond to specific levels of Shh
signaling (Ericson et al., 1997b), being repressed near the floor
plate by high levels of SHH and activated in the intermediate
neural tube at lower levels of SHH. Dorsally, Pax6 is expressed
at reduced levels, except in the roofplate, where no protein is
detectable at the stages examined here (Fig. 1). Studies in
neural explants suggest that the reduced expression of Pax6 in
dorsal tissues might be mediated by activin signaling (Pituello
et al., 1995).
We have found that BMPs regulate Pax6 expression, and that
levels of BMP signaling activity may also play a role in setting
expression boundaries of this gene. In embryos with the highest
levels of BMP signaling (those electroporated with activated
BMPR-Ib), expression of Pax6 was silenced in all cells
expressing the construct (Fig. 1J). This suggests that high
levels of BMP signaling may also be responsible for the
repression of Pax6 expression in roofplate cells. In embryos
electroporated with activated BMPR-Ia (which generates lower
levels of BMP signaling activity) or low concentrations of
BMPR-Ib, Pax6 expression was retained in transfected cells,
but cells in the intermediate region of the neural tube had
reduced levels of Pax6 expression (Fig. 1K,L). In samples with
a moderate number of transfected cells, the normal dorsal
border of high-level Pax6 expression is shifted ventrally
(arrowhead in Fig. 1K). In more thoroughly transfected
embryos, Pax6 was expressed at a uniformly low level, similar
to that normally seen in dorsal cells (Fig. 1L). These data
suggest that BMPs may act to set the dorsal border of the highlevel Pax6 expression.
In the above samples, as well as virally infected samples, a
third effect of BMP signaling on Pax6 expression was apparent.
As shown in Fig. 1M, cells ventral to the high-level Pax6
expression domain expressed increased levels of Pax6 protein
in response to BMP signaling. The reduced expression of Pax6
in this region is thought to be necessary for the proper
generation of motoneurons (Briscoe et al., 2000); we have
found that motoneuron populations are reduced in embryos
expressing activated BMPR, consistent with the results of
Basler et al. (Basler et al., 1993). Thus, it appears that levels
of BMP signaling regulate many borders of expression of Pax6
in the spinal neural tube.
BMP signaling activates dorsal regulators and
represses intermediate regulators
BMP signaling activates Msx expression
The homeobox proteins of the Msx gene family are also
expressed early in neural tube development. In many tissues,
BMP signaling activates Msx expression (Bei and Maas, 1998;
Graham et al., 1994; Maeda et al., 1997; Pizette and
Niswander, 1999; Suzuki et al., 1997), leading us to examine
the expression of these genes in samples expressing activated
BMPR. In mice, the Msx gene family consists of three
members. In the mouse spinal neural tube, Msx1 and Msx2 are
expressed in the roofplate and its overlying mesenchyme and
ectoderm, beginning at times similar to the Pax genes. Msx3
is expressed in a broader region that encompasses the dorsal
third of the neural tube (Shimeld et al., 1996; Wang et al.,
1996). In the chicken spinal neural tube, Msx protein
expression follows a similar time course, but there are
differences in expression patterns. Msx2 RNA is expressed in
the roofplate (Fig. 2A), while Msx1 RNA is expressed in a
manner reminiscent of murine Msx3 (Chan-Thomas et al.,
1993; Liem et al., 1995).
Previous reports have shown that BMPs can induce Msx
expression in cultured neural tube explants (Liem et al., 1995;
Shimeld et al., 1996). Fig. 2A-D shows that activated BMPR
causes a similar activation in the intact neural tube.
Electroporation of activated BMPR results in expression of
Msx2 in all regions of the neural tube, including ventral cells
(Fig. 2B). Although only cells of the roofplate normally express
Msx2, the ectopic expression does not appear to reflect the
BMP regulation of neural tube patterning 2463
Fig. 2. BMP signaling regulates the expression of homeobox genes in the neural tube. (A-D) Expression of Msx genes is upregulated by BMP
signaling. (A,B) Msx2 RNA expression. A stage 24 wild-type sample. (B) Expanded Msx2 expression following electroporation of activated
BMPR-Ib (stage 23). (C,D) Msx1 RNA expression. (C) Stage 24 wild-type embryo. (D) Upregulation of Msx1 expression in an electroporated
embryo expressing activated BMPR-Ib (stage 23). Expression is detected in the ventral neural tube and at higher levels within the endogenous
expression domain. (E-H) Dbx protein expression is repressed by BMP signaling. (E,F) Dbx1 protein expression. (E) Stage 23 wild-type
sample. (F) Cells expressing activated BMPR cease to express Dbx1 (stage 23, viral infection; arrowhead denotes region of BMP-driven
repression). Inset shows viral infection in green; the absence of Dbx1 correlates with viral infection. (G,H) Dbx2 protein expression. (G) Stage
24 wild-type sample. (H) Repression of Dbx2 expression in a stage 23 sample expressing activated BMPR-Ib following viral infection.
Arrowhead indicates a group of cells that no longer express Dbx2. (I-L) Differentiated cell types of the intermediate neural tube are altered in
response to BMP signaling. (I,J) En1 protein expression, which marks the interneurons derived from the ventral-most Dbx2-expressing cells.
(I) Wild-type expression in a stage 24 sample. (J) Arrowhead indicates that this population is greatly reduced in samples expressing activated
BMPR (stage 24, electroporation). Inset shows that the absence of En1 expression correlates with extensive transfection (marked by GFP).
(K,L) Evx protein expression, which marks the interneurons derived from Dbx1+, Dbx2+, Pax7– cells. (K) Wild-type expression in a stage 24
sample. (L) Arrowhead indicates that this population is greatly reduced in samples expressing activated BMPR (stage 24, electroporation). GFP
fluorescence indicates extensive transfection of cells in the region that would normally express Evx.
generation of ectopic roofplate tissue, as cells within these
regions continue to express markers of other neural fates (i.e.
LH2A/B, data not shown), and display normal morphology and
migration. Msx1 is also activated in cells ventral to its normal
border of expression (Fig. 2D). In cells where Msx1 is normally
expressed, activated BMPR appears to increase the levels of
Msx1 transcript, as in situ hybridization analysis revealed groups
of cells with darker staining than normal within the dorsal third
of the neural tube (Fig. 2D). Similar results were obtained with
an antibody that detects both Msx1 and Msx2 (Fig. 3A,B and
data not shown). In the presence of activated BMPR, high levels
of Msx protein detected outside of the roofplate are likely to
reflect both the activation of Msx2 and increased or ectopic
Msx1 expression. Combined with the activation of Pax7
described above, our results indicate that markers of dorsal cell
fates can be activated in ventral neural cells by BMP signaling.
BMP signaling represses Dbx protein expression
The Dbx homeodomain proteins are expressed in nested
domains in the intermediate region of the neural tube (Pierani
et al., 1999) (chicken Dbx2 is also known as ChoxE) and
are necessary for the development of specific classes of
interneurons. In explant experiments, their expression is
activated by retinoic acid and low concentrations of SHH and
repressed by BMPs, suggesting a similar regulation in the
neural tube (Pierani et al., 1999). We have found that BMP
signaling represses Dbx protein expression in intact neural
tissue. Fig. 2F,H show samples infected with activated BMPR;
2464 J. R. Timmer, C. Wang and L. Niswander
Fig. 3. Msx1 mediates the repression of Dbx2
by BMPs. (A,B) Dbx2 and Msx1 expression
domains are juxtaposed. (A) Expression of
Dbx2 and Msx1 in a stage 24 wild-type sample.
Confocal microscopy reveals that cells do not
co-express these proteins. (B) The mutually
exclusive expression of these proteins is retained
in samples expressing activated BMPR (viral
infection, stage 24). Arrowhead indicates cells
expressing Msx1 ventral to its normal
expression border. Confocal microscopy reveals
that these cells do not express Dbx2. (C-E) Misexpression of Dbx2 does not affect Msx1
expression. Samples were generated by
electroporation of viral DNA that drives Dbx2
expression and analyzed at stage 23. Brackets
indicate a region of cells expressing Dbx2
within the normal Msx1 domain; bars represent
endogenous Dbx2 expression domain. Virally
infected cells express Dbx2 at levels equal to or
higher than those in the endogenous domain.
(C) Expression of Msx1 is unaltered by ectopic
expression Dbx2. Co-expressing cells are
apparent in D; E shows that many cells in the
endogenous Msx1 domain are expressing Dbx2.
(F,G) Mis-expression of Msx1 represses Dbx2
expression. Embryos were injected with an
Msx1-expressing virus and analyzed at stage 24.
Arrowhead denotes a region of ectopic Msx1
expression. (F) Gaps in the Dbx2 expression
domain are apparent in the Msx1-infected
samples. (G,H) These gaps correspond precisely
with those cells that ectopically express Msx1.
arrowheads indicate regions of reduced Dbx1 and Dbx2 protein
expression. These data show that BMP signaling represses the
expression of both of these proteins and suggests that it
normally acts to set the dorsal border of their expression
domains.
BMP signaling alters the generation of intermediate cell
types
The BMP regulation of homeobox genes shown above
suggested that embryos expressing activated BMPR would
have reduced populations of some classes of intermediate
interneurons. Dbx2+ cells ventral to the Pax7 expression
boundary generate two types of interneurons: those cells that
express Dbx1 generate Evx1-expressing neurons, while the
more ventral, Dbx1– population generates neurons that express
En1 (Briscoe et al., 2000; Pierani et al., 1999). We have found
that the generation of both Evx1- and En1-expressing
interneurons is severely reduced in embryos that express
activated BMPR (Fig. 2J,L). It is not clear whether the primary
cause of their reduction is the loss of Dbx protein expression
or the expanded expression of Pax7.
Msx1 mediates the BMP driven repression of Dbx2
We noted that the ventral boundary of Msx1 expression
corresponded to the dorsal boundary of Dbx2 expression.
Confocal microscopy revealed that these proteins were
expressed in a mutually exclusive manner (Fig. 3A). This
relationship was retained in embryos where Msx1 expression
was expanded because of the expression of activated BMPR
(arrowhead in Fig. 3B), suggesting a regulatory interaction
between these genes. To examine potential interactions
between Msx1 and Dbx2, we used RCAS vectors to express
these genes in the developing neural tube. Fig. 3C-E shows
that, even in the presence of high levels of Dbx2 (compare
ectopic expression with endogenous expression), Msx1
expression remains uniform in the dorsal third of the neural
tube, indicating that Dbx2 is not capable of regulating Msx1
expression. By contrast, when Msx1 expression occurs in cells
within the normal domain of Dbx2 expression, gaps appear in
the otherwise uniform field of Dbx2 expression (Fig. 3F-H).
Fig. 3F,G (note arrowheads) shows that those cells that fail to
express Dbx2 correspond precisely to those cells ectopically
expressing Msx1. These data strongly suggest that BMP
signaling acts through Msx1 to repress Dbx2 expression.
Within the dorsal neural tube, the expression of
developmental regulators responds to discrete
levels of BMP signaling
Several genes of the bHLH family of transcription factors are
expressed in well-defined domains within the Msx1 expression
domain in the dorsal third of the neural tube, including the
acheate-scute homolog Cash1 (Guillemot and Joyner, 1993),
the chicken atonal homolog 1, Cath1 (Lee et al., 1998),
neurogenin 1 (Ngn1), and neurogenin 2 (Ngn2) (Ma et al.,
1996; Sommer et al., 1996). We have observed changes in the
expression of all of these genes in response to BMP signaling;
BMP regulation of neural tube patterning 2465
Fig. 4. Expression of activated BMPR alters the expression of bHLH proteins in the dorsal neural tube. (A,B) Cath1 expression expands
ventrally in response to BMP signaling. (A) Wild-type expression of Cath1 protein occurs in cells immediately ventral to the roofplate (stage 23
sample). (B) Expression of Cath1 expands dramatically in response to expression of activated BMPR (stage 24, viral infection). Note that the
expanded expression remains restricted to dorsal neural tissue. Inset shows viral antigen expression, indicating infected cells; expanded Cath1
correlates with areas of extensive viral infection. (C,D) Cash1 expression is repressed by BMP signaling. (C) Wild-type expression of Cash1
protein includes a broad band of cells in the dorsal neural tube (stage 24). (D) The Cash1 domain is reduced in samples expressing activated
BMPR (stage 23 viral infection). (E,F) Dorsal Ngn2 expression is reduced by BMP signaling. (E) Wild-type expression of Ngn2 RNA is found
in ventricular and subventricular cells in the dorsal half of the neural tube, as well as a large population of ventral cells (stage 24; arrowhead
indicates dorsal expression domain). (F) Expression of Ngn2 in most dorsal cells is extinguished by the expression of activated BMPR (stage 24
electroporation); ventral expression remains unaffected. (G,H) Dorsal expression of Ngn1 is repressed by BMP signaling. (G) At stage 24,
Ngn1 RNA is expressed by cells of the dorsal root ganglia (DRG), proliferating ventral cells and a narrow band of cells in the dorsal neural tube
(arrowhead). (H) Ngn1 expression in the dorsal neural tube is absent in samples expressing activated BMPR (stage 24 electroporation), while
ventral and DRG expression remains normal.
combined with the results of others, these results indicate that
BMPs act at specific levels of activity to pattern the dorsal
neural tube.
BMP signaling activates cath1 throughout the dorsal
neural tube
The Cath1 gene is expressed by the cells of the ventricular zone
that reside between the roofplate and an Ngn1-expressing
dorsal cell population (Lee et al., 1998). Cath1 protein
expression has been shown be activated by BMPs in explants
and partially dependent upon the BMP relative GDF7 in mice.
In samples expressing activated BMPR, Cath1 is activated in
most cells of the dorsal neural tube (Fig. 4B); no expression
was detected in ventral cells. This result suggests that the
activated BMPR converted dorsal neural cells to a more dorsal
fate, marked by Cath1 expression.
BMP-induced expansion of Cath1 correlates with the
repression of other bHLH genes
The Cash1 protein is expressed in a broad band of cells in the
dorsal neural tube with a dorsal border several cell diameters
ventral to the roofplate; levels of Cash1 protein expression
appear to vary within this domain (Fig. 4C). In experiments
where the roof plate was ablated, Lee et al. (Lee et al., 2000)
found that the mouse homolog of this gene was expressed
uniformly throughout the dorsal third of the neural tube,
including the roofplate. This led these authors to conclude that
BMP-mediated repression was responsible for setting the
dorsal expression border of this gene. Our data support this
conclusion; in response to BMP signaling, we have found that
dorsal expression of Cash1 is repressed (Fig. 4D), although
some expression was always retained near its ventral border.
Combined with the previous results, these data imply that the
dorsal border of Cash1 expression is set at a threshold of BMP
signaling activity.
The bHLH gene Ngn2 is expressed by most ventral cells. In
dorsal cells, Ngn2 transcripts are detected in a narrow band of
cells in the ventricular zone, similar to those that express Ngn1,
as well as a larger population of subventricular cells (Perez et
al., 1999). Fig. 4F shows that BMP signaling represses the
dorsal expression of Ngn2. Some dorsal cells retained normal
expression of Ngn2 in all samples examined. As fluorescence
from the co-electroporated GFP does not survive in situ
analysis, it is not clear whether this reflects a tolerance of high
levels of BMP signaling by a subdomain of Ngn2-expressing
cells or the lack of uniform activated BMPR expression in the
cells of the dorsal neural tube. The related gene Ngn1 is also
expressed broadly in ventral cells; additionally, ngn1 transcripts
2466 J. R. Timmer, C. Wang and L. Niswander
are expressed in a narrow band of cells near the roofplate (Fig.
4G). As shown in Fig. 4H, expression of activated BMPR also
resulted in the loss of the dorsal Ngn1 expression domain; as
with Ngn2, ventral expression was unaffected.
Ngn1 expression is regulated by thresholds of BMP
signaling
In the roofplate ablation experiments (Lee et al., 2000),
absence of BMP signaling resulted in the loss of the dorsal
domain of Ngn1. As Ngn1 both requires BMP signaling for its
expression and is repressed by high levels of BMP signaling,
BMPs may regulate Ngn1 expression at specific thresholds of
activity. In order to test this, we used a construct that expresses
extremely low levels of activated BMPR (see Materials and
Methods for a description). If BMP signaling is also capable
of activating Ngn1, the low level of BMP signaling generated
by this construct, when combined with endogenous BMP
signaling from the roofplate, should be capable of activating
Ngn1 transcription in cells ventral to its endogenous expression
domain. As shown in Fig. 5A, these samples frequently had
ventral expansion of Ngn1 expression on the electroporated
side of the neural tube. This expansion correlated with an
increase in the generation of terminally differentiated neurons
that arise from cells near the endogenous ngn1 domain (Fig.
5D, discussed below). These results suggest that genes within
the dorsal neural tube respond to specific thresholds of BMP
signaling.
BMP regulation of dorsal patterning genes controls
the generation of populations of differentiated
neurons
Previous work has identified several populations of
interneurons that are derived from the cells of the dorsal
neural tube. Cath1-expressing cells give rise to terminally
differentiated cells that express either LH2A or LH2B and
migrate to ventral positions (Helms and Johnson, 1998; Liem
et al., 1997). More ventral cell types generate differentiated
cells that express Islet1 (Isl1) or Lim1 and Lim2, which also
migrate to ventral positions in the neural tube (Fig. 5D,G). We
have found that altering levels of BMP signaling dramatically
alters the generation of these classes of dorsal neurons.
As expression of ventral markers is replaced by Cath1
expression in samples expressing activated BMPR, we sought
to determine if there was a corresponding increase in the
generation of Cath1 derivatives, which express LH2A or LH2B.
Normal expression of LH2A/B is shown in Fig. 5B. As would
be predicted, in samples expressing activated BMPR, cells
expressing LH2A/B develop throughout the dorsal neural tube
(arrowhead in Fig. 5C). As with Cath1, the expanded expression
is limited to cells in the dorsal third of the neural tube.
Cells expressing Lim1/2 are generated at various locations
along the dorsoventral axis of the neural tube. In addition to
ventral populations, terminally differentiated neurons arising
from two dorsal locations express this marker (bars in Fig. 5D).
Expression of activated BMPR in dorsal cells frequently
reduced and occasionally eliminated these dorsal populations
(arrowhead in Fig. 5E). Cells generated at more ventral
locations continue to express these proteins, and some Lim1/2+
cells were generated at ectopic, ventral positions within the
ventricular zone of undifferentiated cells. The significance of
the ectopic generation of Lim1/2 cells is unclear at this time.
The Isl1 protein is expressed by a variety of neural cell types,
including neural crest derivatives in the dorsal root ganglia,
differentiated motoneurons and a population of dorsal
interneurons (Fig. 5G, dorsal interneurons indicated by a bar)
(Tsuchida et al., 1994). In samples electroporated with activated
BMPR, this population was reduced or absent, depending on
the extent of activated BMPR expression (arrowhead in Fig.
5H). As Isl1 cells arise from cells near where Ngn1 is expressed,
we decided to examine Isl1 expression in samples
electroporated with the low-level activated BMPR expression
construct that was shown to expand Ngn1 expression. In many
samples expressing this construct, the number of Isl1expressing dorsal cells was increased. In addition, these cells
arise from a broader progenitor domain (bracket in Fig. 5F).
Taken together, these data indicate that BMP-driven changes in
progenitor cell markers correlate with changes in differentiated
cell populations.
These results show that BMP driven changes in gene
expression in undifferentiated neurons reflect a change in the
identity of a cell. Thus, within the Msx1 expression domain,
BMP signaling acts to dorsalize both immature and
differentiated neurons. By generating specific cell types at
thresholds of activity, the BMPs ultimately regulate the
diversity and numbers of dorsal neurons.
DISCUSSION
Our data suggest that BMPs provide patterning information in
the dorsal and intermediate regions of the neural tube. In the
dorsal neural tube, BMP signaling regulates a series of bHLH
proteins in order to generate several distinct populations of
neural progenitor cells; in intermediate cells, BMP signaling
performs the same function via the regulation of homeodomain
proteins. In both cases, the generation of these populations is
necessary for generating a diverse population of differentiated
neurons. BMPs appear to act in a cell autonomous manner in
performing this function; although we cannot rule out a role
for secondary signals, the consistent correlation of patterning
alterations with transfected cells and absence of changes in
neighboring cells argues against a significant role for other
signals. The data also indicate that BMPs act instructively, as
changes in patterning were not accompanied by regional
changes in apoptosis or proliferation.
We have seen only quantitative differences between
constructs that express BMPR-Ia and BMPR-Ib. This differs
from the results of Panchision et al. (Panchision et al., 2001),
who expressed similar constructs in a mouse transgenic
system. Although data from both agree on a role for BMPs in
dorsalizing neural tissue, Panchision et. al. suggest distinct
roles for the two BMP receptors. Their data, however, were
generated partly in mouse transgenics and partly in cultured
neural stem cells. There are likely to be significant differences
in the cells used, timing of construct expression, and levels of
construct expression between these systems and the in ovo
electroporation we used, which may account for the different
results.
Our data, however, are consistent with results obtained
with in vitro explants of neural tissue, but expand on them in
significant ways. It is clear that other signaling pathways are
necessary for proper development of the neural tube; neural
BMP regulation of neural tube patterning 2467
Fig. 5. BMP signaling regulates the generation of terminally differentiated dorsal interneurons. (A) Ngn1 RNA expression is activated at a
threshold of BMP signaling. Ngn1 is activated by low levels of BMP signaling. A construct that weakly expresses activated BMPR-Ib (EGFP
vector) results in an increase in Ngn1-expressing cells, apparently by activating it in cells ventral to its normal domain (arrowhead, stage 24).
(B,C) LH2A/B-expressing cells are generated throughout the dorsal neural tube in response to high levels of BMP signaling. (B) In stage 23
wild-type samples, LH2A/B-expressing cells are generated by those cells that express Cath1 and migrate ventrally. (C) In response to high level
BMP signaling, LH2A/B+ cells are generated throughout the dorsal neural tube (arrowhead, stage 24). Inset shows co-localization of GFP and
LH2A/B expression on the transfected side of the neural tube. (D,E) High levels of BMP signaling block the development of dorsal Lim1/2+
cells. (D) In wild-type samples, Lim1/2+ cells are produced in two locations within the dorsal neural tube (bars; stage 23). (E) Expression of
activated BMPR reduces or eliminates the generation of the two dorsal populations of Lim1/2-expressing cells (arrowhead denotes normal
region of their generation, stage 23). Inset shows GFP expression, indicating transfected cells. Note that suppression of Lim1/2 expression is
specific to dorsal cells, as intermediate expression continues in transfected cells. (F-H) Isl1-expressing cells are formed within a narrow range
of BMP signaling activity. (F) Samples with slight increases in BMP signaling (generated with the same vector as in A, EGFP-activated BMPRIb) generate Isl1-expressing cells over a broader region of the dorsal neural tube (bracketed; bar indicates endogenous domain, late stage 24).
Inset shows GFP label of transfected cells. Note that Isl1 expansion is restricted to the region near its normal expression domain, despite
extensive transfection. (G) In stage 24 wild-type samples, Isl1 expression marks a small population of dorsal interneurons (bar) as well as the
DRG and motoneurons.(H) High levels of BMP signaling, however, abolish the generation of Isl1+ interneurons (arrowhead, stage24). Inset
shows that the absence of Isl1 is correlated with the presence of transfected cells marked by GFP.
explants are likely to have altered expression of the
components of pathways such as Wnt and Shh (Chiang et al.,
1996; Dickinson et al., 1994; Ericson et al., 1995). Additional
evidence has suggested that signals arising from non-neural
tissues, such as the dorsal ectoderm and somites, are also
necessary for neural patterning (Bertrand et al., 2000; Liem
et al., 2000; Pierani et al., 1999). Moreover, examination of
the effects of BMPs in intact tissue has allowed us to identify
regulatory interactions (i.e. Msx1/Dbx2) and regionalspecific responses to BMP signaling (i.e. dorsal-specific
changes in bHLH expression). Thus, these experiments
present a clearer view of the role of BMP signaling in its
proper context.
BMPs act in conjunction with Shh to set Pax gene
expression borders
One of the earliest signs of distinct regional identity in spinal
neural tissue is the expression of Pax genes, specifically Pax6
and Pax3/7 (Liem et al., 1995; Pituello et al., 1995). Our data
indicate that BMP signaling plays a role in setting the expression
boundaries of these Pax genes. We have found that BMP
signaling promotes the expression of Pax7 in ventral tissue, both
shifting its expression boundary ventrally and causing ectopic
Pax7 expression. BMP signaling also appears to regulate Pax6
in a level-dependent manner. High levels of BMP signaling
eliminate Pax6 expression; this repression may indicate a role
for BMPs in eliminating Pax6 expression in the roofplate.
2468 J. R. Timmer, C. Wang and L. Niswander
Fig. 6. BMP signaling regulates many aspects of neural tube
patterning. (A) BMP signaling regulates homeobox gene expression
to define dorsal and intermediate cell fates. In cooperation with SHH
signaling, BMPs set the expression domain boundaries of Pax6 and
Pax7. The border between dorsal and intermediate cell fates, marked
by the dorsal border of high level Pax6 expression is refined by the
BMP-mediated activation of Msx1, which represses Dbx2
expression. (B) BMP signaling regulates the dorsal expression of
bHLH proteins along a gradient of activity. bHLH protein expression
boundaries are set by thresholds of BMP signaling. bHLH expression
domains give rise to a limited number of types of terminally
differentiated neurons. (C) BMP signaling promotes a diversity of
intermediate cell fates. BMP regulation of Pax7 sets a dorsal limit on the generation of Evx1-expressing neurons. BMP regulation of the dorsal
border of Dbx1-expressing cells may help divide the Pax2+, Lim1/2+ cells into two distinct progenitor populations.
Moderate levels of BMP signaling shift the border of the
intermediate domain of high Pax6 expression ventrally, while in
samples with extensive construct expression, the high level
expression domain is eliminated entirely. Finally, we have found
that low levels of BMP signaling are capable of expanding the
intermediate domain of Pax6 expression ventrally.
Previous data had indicated that the Pax genes were also
regulated by SHH signaling. In the case of Pax7, the absence
of expression in ventral tissues is caused by a SHH-mediated
repression (Goulding et al., 1993; Hynes et al., 2000; Liem et
al., 1995). Pax6 expression also depends on SHH; high levels
of SHH repress Pax6 expression in ventral cells, while lower
levels activate Pax6 in the intermediate region of the neural
tube (Ericson et al., 1997b). Thus, the majority of Pax
expression domain boundaries appear to be set by the
combined action of the BMP and SHH signaling pathways (see
Fig. 6A). The regulation of Shh-responsive genes by BMPs
shown here does not reflect BMP regulation of Shh expression,
as Shh transcript remained unaltered in virally infected samples
(data not shown). It is possible be that these signaling pathways
interact downstream of ligand expression, as it has been found
that BMP activity can influence the reception of Shh signaling
(Liem et al., 2000).
BMPs coordinate the expression of two homeobox
proteins and help to generate distinct dorsal and
intermediate cell populations
The dorsal border of Pax6 expression is not a sharp boundary
such as those that frequently separate distinct cell types,
suggesting that other factors may contribute to the delineation
of dorsal and intermediate cells. We have found that a sharp
border between dorsal and intermediate cell types is generated
at this location by the BMP-regulated expression of two
homeobox proteins (Msx1 and Dbx2) (Fig. 6A): the dorsal
border of Pax6 expression corresponds to both the ventral
border of Msx1 and the dorsal border of Dbx2. We have shown
that the BMP-mediated activation of Msx1 expression sets a
dorsal boundary on Dbx2 expression. As the expression of
Dbx2 appears to be necessary for the proper development of
several intermediate cell types (Pierani et al., 1999), BMP
regulation of Msx1 limits the population of cells that can serve
as progenitors for intermediate neurons. In contrast to Dbx2,
expression of Msx1 is not sufficient to generate dorsal cell
types (compare Fig. 2 with Fig. 4). Thus, Msx1 may only be
one of several competence factors that promote dorsal identity.
A requirement for multiple factors to generate dorsal
competence may explain why misexpression and loss-of-
BMP regulation of neural tube patterning 2469
function mutations in many of the genes expressed in
restricted patterns in the neural tube have limited phenotypes
in this tissue (Mansouri and Gruss, 1998; Tremblay et al.,
1996). Nevertheless, the different responses of bHLH proteins
in dorsal and ventral neural tissue suggest that these tissues
have been assigned distinct developmental potentials.
BMPs contribute to the generation of a diversity of
intermediate neural cell types
In addition to limiting the population of cells that are
competent to generate intermediate cell types, BMPs regulate
the types of interneurons generated by this Dbx2+ population.
Within this cell population, specific cell types are generated by
populations defined by overlapping expression of homeobox
proteins (Briscoe et al., 2000; Mansouri and Gruss, 1998;
Pierani et al., 1999). Cells expressing both Dbx2 and Pax7 give
rise to interneurons expressing Pax2 and Lim1/2 (Burrill et al.,
1997). Pax7 has also been shown to help set a dorsal limit on
the generation of Evx1-expressing interneurons (Mansouri and
Gruss, 1998; Pierani et al., 1999). Thus, by regulating the
ventral border of Pax7 expression, BMPs promote the
generation of the dorsal cells at the expense of more ventral
intermediate cell types, such as Evx1- and En1-expressing
interneurons. Accordingly, we have seen a reduction in the
generation of these ventral cell fates in embryos where BMP
signaling is activated in intermediate cells.
As shown in Fig. 6C, Dbx2+ cells ventral to the Pax7
expression border give rise to two cell populations, determined
by the presence or absence of the homeobox protein Dbx1. As
Dbx1 also has an expression border within the Pax7 expression
domain, it is possible that Dbx1 performs a similar function in
generating two distinct interneuron populations within the
Pax2-expressing population. If this were the case, BMPmediated regulation of Dbx1 (Pierani et al., 1999) would also
be necessary for the proper patterning of other intermediate cell
types. We are unaware of the expression of any protein that
marks distinct populations within these cells, however, so we
have been unable to test this.
BMP ligands control the generation of dorsal
interneurons by regulating gene expression at
distinct thresholds of activity
In addition to their role in promoting the formation of an Msx1expressing dorsal progenitor pool, BMPs also act to subdivide
this domain into discrete cell populations (see Fig. 6D). Several
of the cell types generated by this region depend upon the
expression of bHLH proteins (Gowan et al., 2001) that we have
been found to be regulated by BMPs. We have found that high
levels of BMP signaling activity promote the expression of the
most dorsal bHLH protein, Cath1. The expanded domain of
Cath1 expression, however, remains restricted to the dorsal
neural tube. The broad expression of Cath1 correlates with the
repression of other regulators expressed in this region, such as
the neurogenins and Cash1. As with Cath1, this regulation is
specific to dorsal neural cells; neurogenin expression in ventral
cells appears to be unaltered. The restriction of these responses
to dorsal cells is consistent with this region having been
previously assigned a distinct developmental identity and
potential. It is also consistent with findings that the dorsal and
ventral regulatory sequences of the neurogenins are physically
separated (Simmons et al., 2001).
Our data, when viewed in conjunction with previous data
from others, indicate that thresholds of BMP signaling set the
expression boundaries of dorsal regulators. We have found that
high levels of BMP signaling repress Cash1 in its most dorsal
domain, although expression is retained in more ventral cells
of this domain. In experiments where the roofplate was
genetically ablated and expression of its BMPs abolished,
Cash1 expression was expanded dorsally (Lee et al., 2000;
Millonig et al., 2000). Thus, it appears that BMP signaling sets
the dorsal border of Cash1 expression at a precise level of
activity, while expression in more ventral cells is independent
of BMP activity. By contrast, dorsal Ngn1 expression is absent
both in embryos with high levels of BMP activity and those in
which the roofplate is absent and BMPs are not expressed (Lee
et al., 2000). This suggests that the dorsal cells that express
Ngn1 are formed within a limited range of BMP activity. We
have confirmed this suggestion by showing that Ngn1
expression is broadly activated in the dorsal neural tube by low
levels of BMP signaling. These results indicate that the BMPs
collectively act to regulate genes along a gradient of activity,
possibly behaving as morphogens to pattern cells within the
dorsal neural tube (Fig. 6D). This function would be consistent
with other descriptions of BMPs and other TGFβ superfamily
members acting as morphogens during embryonic
development (Dale and Jones, 1999; McDowell and Gurdon,
1999; Podos and Ferguson, 1999). The recent finding that the
bHLH genes expressed in this region inter-regulate (Gowan et
al., 2001) indicates the BMP activity may only have to set the
boundary of a limited number of gene(s) in order to generate
several distinct cell populations.
The regulation of the dorsally expressed genes by BMP
signaling has significant consequences for the generation of
differentiated dorsal interneurons. The expanded expression of
Cath1 drives the generation of LH2A/B+ cells throughout the
dorsal third of the neural tube. The expansion of Lh2A/B+
interneurons appears to come at the expense of other cell fates,
as the generation of other classes of dorsal interneurons, marked
by Isl1 or Lim1/2, is reduced or eliminated. Thus, the presence
of high levels of BMP signaling throughout the dorsal third of
the neural tube appears to generate a uniform pool of
progenitors and a single type of differentiated neuron in this
region. Small increases in BMP signaling in dorsal cells,
however, can expand the generation of other cell types in this
region (Ngn1 and Isl1, see Fig. 5A,F). This reinforces the
suggestion that a gradient of BMP signaling activity is essential
for the generation of a diversity of dorsal interneurons (Fig. 6D).
BMP signaling and transcription factor codes
Previous work has shown that homeobox containing proteins
play a significant role in the generation of differentiated neural
cell types (Briscoe et al., 2000). A homeobox ‘code’, generated
by overlapping and exclusive domains of expression, provides
the positional information necessary for generating many
classes of neurons at precise locations in the ventral neural
tube. We have shown that BMPs contribute to the generation
of the homeobox code. BMP regulation of Pax6 expression sets
a dorsal limit on the population of cells that give rise to
motoneurons. By regulating the ventral boundary of Pax7
expression, BMPs act to limit the ability of cells to adopt
ventral fates. BMPs also act to set a dorsal boundary to Dbx2derived intermediate cell fates via activation of the homeobox
2470 J. R. Timmer, C. Wang and L. Niswander
protein Msx1. We also expect that the BMP-regulated dorsal
border of Dbx1 expression will play a similar role in
subdividing the intermediate neural tube as its ventral border
performs (Pierani et al., 1999).
It remains to be determined whether Msx1 plays a role in
directly providing positional information to differentiating
neurons. It is clear, however, that several types of differentiated
neurons arise from the Msx1 expression domain. The division
of this domain into distinct progenitor populations depends on
the expression of bHLH proteins (Gowan et al., 2001; Lee et
al., 1998). Consistent with this, we have found that alterations
in the differentiated cells generated by cells within the Msx1
expression domain correlate with changes in the expression of
bHLH proteins (Cash1, Ngn1, Ngn2 and Cath1) expressed by
undifferentiated cells in this region.
Restricted expression of bHLH proteins, however, does not
appear to be the only mechanism for patterning the dorsal
neural tube. The cells that express Cash1 appear to give rise to
at least three types of differentiated neurons (Isl1+, a second
Lim1/2+ population and Lmx1b+), but no currently identified
bHLH protein appears to further subdivide the Cash1
expression domain. In addition, any direct relationship between
bHLH protein expression and cell fate is restricted to dorsal
cells, as most of these bHLH proteins are also broadly
expressed in ventral cells. Thus, the activity of any bHLH code
may ultimately depend on the homeobox proteins that define
the dorsal cell population in which they function.
Modulation of BMP responses
It is clear from these data that the effectors of BMP signaling
must interact with other factors to generate the responses seen
here. The activation of Msx protein expression by BMP
signaling appears to be a general downstream response, as it is
seen in a variety of tissues (Pizette and Niswander, 1999; Streit
and Stern, 1999; Wang et al., 1999; Yamamoto et al., 2000).
Within the neural tube, however, the responses of other genes
are more finely regulated. The response of all other markers
examined here was restricted to neural cells, even when viral
infection spread to surrounding tissues. Within the developing
neural tube, responses to BMP signaling were frequently
restricted to specific regions (e.g. Cath1 activation in the dorsal
neural tube). Thus, at the transcriptional level, the effectors of
BMP signaling must be interacting with other regulators of
transcription or other signaling pathways.
Previous data have suggested that BMPs may cooperate with
the activin members of the TGFβ superfamily, which are also
expressed by the roofplate. Experiments using neural explants
had previously indicated that several of the effects seen here
might also be mediated by activin signaling (Liem et al., 1997;
Pituello et al., 1995). We have found, however, that expression
of an activated activin receptor (Chang et al., 1997) in vivo is
incapable of generating the alterations of gene expression seen
here. Although activin signaling expanded the expression of
Isl1, the expression of many of the BMP-responsive genes
examined here (including Msx, Pax6, Cash1 and Lh2a/b) was
unaltered in embryos expressing this construct (J. T. and L. N.,
unpublished). Thus, these responses appear to be specific to
BMP-mediated signaling.
In addition to their regulation of patterning within the neural
tube, it is worthwhile noting that BMPs have also been
implicated in earlier stages of neurogenesis, including the
generation of neural progenitors from the neurectoderm and the
formation of the roofplate and neural crest (Baker and BronnerFraser, 1997; Liem et al., 1997; Liem et al., 1995), as well
as later events, such as the directing the migration of
differentiated dorsal interneurons (Augsburger et al., 1999).
Many of these processes overlap temporally (e.g. the
generation of the roofplate and neural crest occur while the
generation of patterning information is in progress), and thus
should be viewed as different responses to the continual
presence of BMP signaling. How the response to BMP
signaling is redirected to different processes or used for several
processes simultaneously also provides an interesting avenue
for future study.
Summary
A model for the action of BMP signaling in patterning the
neural tube is depicted in Fig. 6. Initially, BMPs act in
conjunction with Shh signaling to set expression boundaries
for the Pax genes. The Pax6 expression boundary that marks
the border between dorsal and intermediate cells is refined by
the BMP-mediated activation of Msx1 in dorsal cells, which
in turn represses the intermediate marker, Dbx2. This
generates two pools of progenitor cells (intermediate and
dorsal) with distinct developmental potentials. In intermediate
cells, BMPs contribute to the generation of overlapping
patterns of homeobox protein expression; their regulation of
Dbx1, Dbx2 and Pax7 generates at least three distinct
populations of progenitor cells. In dorsal cells, our data
suggest that patterning information is provided in part by
mutually exclusive expression of members of the bHLH
family of proteins. bHLH gene expression is set at specific
thresholds of BMP signaling activity, which ultimately results
in populations of mature neurons differentiating along a
gradient of BMP activity.
The authors acknowledge T. Jessell, J. Johnson, C. Abate-Shen, J.
Eggenschwiler and members of their laboratory for advice, reagents
and critical reading of this manuscript; and Louise Howe, Cathy
Chesnutt and Joan Seoane for assistance with these experiments.
Confocal images were obtained with the help of Katia Manova and
the staff of the Molecular Cytology facility at the Sloan-Kettering
Institute. This work was supported by an NRSA fellowship (C. W.)
an award from the Irma T. Hirschl Trust (L. N.) and by the MSKCC
Cancer Center support grant (CA-08748). L. N. is an assistant
investigator of the Howard Hughes Medical Institute.
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